A 2004 U.S. Department of Energy report entitled “Top value added chemicals from biomass” has identified twelve building block chemicals that can be produced from renewable feedstocks. The twelve sugar-based building block chemicals are 1,4-diacids (succinic, fumaric and maleic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol.
Building block chemicals are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. The twelve building blocks identified by U.S. Department of Energy can be subsequently converted to a number of high-value bio-based chemicals or materials.
During the last few years a number of microorganisms have been created through genetic engineering for the production of industrially useful monomeric building block chemical compounds. Many natural metabolites derived from biological fermentative processes such as dicarboxylic acids, amino acids, and diols have functional groups that are suitable for polymerization and chemical synthesis of industrially useful polymers.
In recent years attention has been focused on reducing the cost of production of industrially useful chemical compounds through biological fermentation. One well known approach for reducing the cost of fermentative production of chemical compounds is to use low-cost minimal medium in place of expensive nutritionally rich medium. For example, the E. coli strain described in U.S. Pat. No. 7,223,567 requires a rich medium supplemented with glucose as the source of carbon for the production of succinic acid. The E. coli strain KJ122 useful for the production of succinic acid described by Jantama et al (2008a and 2008b) and in the PCT Patent Application Publications Nos. WO/2008/021141A2 and WO/2010/115067 is capable of growth on a minimal medium without the need for any expensive ingredients such as yeast extract or tryptone. Another approach that is being attempted to further reduce the cost of fermentative production of chemical compounds is to replace the currently used expensive feedstocks such as dextrose and sucrose with cheaper organic carbon source such as a mixture of six-carbon and five-carbon sugars derived form lignocellulosic biomass through a pretreatment process.
The inventors have discovered a novel method for further reducing the cost of producing specialty chemicals through biological fermentation. This new method for improving the productivity and the yield of succinic acid through a biological fermentation process is based on the observation that the yield and productivity of succinic acid in the biological fermentation process requires the supply of both organic carbon and inorganic carbon sources. A reduction in the cost of production of succinic acid can be achieved by means of supplying the required inorganic carbon in a cost effective manner besides meeting the requirement for organic carbon sources.
As defined in this invention, the term organic carbon refers to the organic feedstocks such as xylose, glucose, glycerol and sucrose useful for the fermentative production of organic acid by the microorganism. The term inorganic carbon refers to the carbon dioxide present in the gas phase of the fermentation chamber and the carbonate and bicarbonate salts added as a component of the fermentation medium.
The importance of the contribution from inorganic carbon towards succinic acid production by microbial catalysts is now well established although the relative contribution of inorganic and organic carbon fractions to the final succinic acid production is not precisely established.
While the transformation of organic carbon into succinic acid is achieved by the modification of the central metabolic pathway including the glycolytic pathway and the tricarboxylic acid cycle within the cell, the incorporation of inorganic carbon into succinic acid requires the participation of carboxylating enzymes. At least four different types of carboxylating enzymes are known to be functional within bacterial cells. The phosphoenol pyruvate carboxylase (PEBcase or PPC) carboxylates phosphoenol pyruvate leading to the formation of oxaloacetic acid. The malic enzyme carboxylates pyruvic acid leading to the formation of malic acid and requires reduced cofactors such as NADH or NADPH. The third carboxylating enzymes known as pyruvate carboxlase (PYC) carboxylates pyruvic acid to produce oxaloacetic acid. The fourth carboxylating enzyme known as phosphoenolpyruvate carboxykinase (PCK) carboxylates phosphoenol pyruvate to oxaloacetate with the production of one molecule of ATP for every molecule of oxaloacetate produced from the carboxylation of a phosphoenol pyruvate molecule. The inorganic carbon assimilated through the carboxylation reactions mediated by one of these four different carboxylating enzymes present within a bacterial cell contributes to the carbon back bone of the succinic acid produced through fermentation process.
The E. coli strains currently in use for the production of succinic acid are reported to have enhanced activity for one or other carboxylating enzymes. U.S. Pat. No. 6,455,284 discloses the use of an exogenous pyruvate carboxylase enzyme for enhancing the production of oxaloacetate-derived chemicals through fermentation. Expression of Rhizobium etli pyruvate carboxylase gene in E. coli cells caused an increased carbon flow towards oxaloacetate in wild type E. coli cells without affecting the glucose uptake rate or the growth rate and restored succinate formation in E. coli phosphoenolpyruvate carboxylase null mutants. Zhang et al (2009) have reported that in KJ122 strain of E. coli due to a mutation in the promoter region, the phosphoenolpyruvate carboxykinase enzyme shows enhanced carboxylation capacity.
Sanchez et al (2005) have reported that the flux to the oxaloacetate pool was increased by overexpressing the enzyme pyruvate carboxylase (PYC) from Lactococcus lactis in E. coli cells. The synthesis of oxaloacetate is a key step towards the synthesis of succinate. In wild-type E. coli phosphoenol pyruvate carboxylase represents the principle anaplerotic reaction to replenish oxaloacetate. Under anaerobic conditions the portion of phosphoenolpyruvate not flowing to oxaloacetate is converted to pyruvate. In strains not expressing the heterologous pyruvate carboxylase, pyruvate was observed to accumulate and succinate yield decreased compared to the strain overexpressing pyruvate carboxylase.
Lin et al (2005) have shown that the highest level of succinate production in E. coli can be achieved by expressing both phosphoenol pyruvate carboxylase from Sorghum vulgare and pyruvate carboxylase from Lactococcus lactis when compared to E. coli strains individually overexpressing either phosphoenol pyruvate carboxylase or pyruvate carboxylase.
As indicated by these studies, all the efforts so far have been focused on increasing the succinic acid production capability by means of effectively utilizing the inorganic carbon already present within the cell. This present invention provides a novel method for enhancing the inorganic carbon uptake by bacterial cells leading to an increase in the concentration of inorganic carbon within the bacterial cell with the ultimate goal of increasing the succinic acid production.
Generally, the inorganic carbon requirement for the fermentative production of succinic acid is supplied either in the form gaseous carbon dioxide or in the form of a carbonate salt such as sodium carbonate, sodium bicarbonate, ammonium carbonate, and ammonium bicarbonate. A number of US patents have disclosed the use of inorganic carbon either to maintain the pH of the culture medium or to maintain the growth rate of the microorganism. For example, U.S. Pat. No. 5,958,744 uses NaHCO3 to neutralize the succinic acid produced by the E. coli strain AFP 111. The sodium bicarbonate addition to the fermentation medium besides maintaining the neutral pH, also serves as a source of inorganic carbon required for the carboxylation reactions within the cell. Andersson (2007) has demonstrated that the use of Na2CO3 as a neutralizing agent is desirable over the use of NH4OH, KOH, and NaOH as neutralizing agents. It has been reported that NH4OH as a neutralizing agent is toxic to E. coli and could cause a decrease in the viability of the cells and the succinate productivity (Andersson et al., 2009). Thus the prior art teaches away from the use of NH4OH as the neutralizing agent in the succinic acid production.
Andersson et al (2007) have disclosed the use of gaseous carbon dioxide in the production of succinic acid using the metabolically engineered E. coli strains AFP 111 and AFP184. These succinic acid producing strains were grown in a medium maintained at pH between 6.6 and 6.7 with the addition of NH4OH as 15% NH3 solution. The anaerobic production phase was initiated by withdrawing the air supply and sparging the culture medium with CO2 at a flow rate of 3 L min−1.
U.S. Pat. No. 5,168,055 discloses that the growth conditions for succinic acid producing Anaerospirillum succiniproducens requires at least about 0.1 atmospheric CO2. The medium can be sparged with CO2 gas. The fermentation can be run in a pressurized reactor which contains CO2 at super atmospheric pressure. The CO2 can be mixed with other gases as long as the gases employed do not interfere with the growth. Carbon dioxide can also be supplied to the fermentation medium by the addition of carbonate or bicarbonate salts which generates CO2 gas under the conditions of the fermentation. For sufficient succinic acid production, the medium should contain dissolved CO2 in equilibrium.
Promising succinic acid producing bacteria Mannheimia succinciproducens and Actinobacillus succinogens have been isolated from bovine rumen. The major gas produced in the rumen of the cattle is CO2 (65.5 mol %). These strains of rumen bacteria are capnophilic (CO2 loving) and produce succinic acid as the major product from various carbon sources under 100% CO2 conditions at pH of 6.0 to 7.5. Genome-scale metabolic flux analysis indicated that CO2 is important for the carboxylation of phosphoenolpyruvate to oxaloacetate, which is converted to succinic acid by the reductive tricarboxylic acid cycle (Lee et al., 2002; Hong et al., 2004; Song and Lee., 2006).
Song et al (2007) have shown that in the capnophilic rumen bacterium M. succiniproducens the production of succinic acid by a carboxylation reaction during fermentation is dependent on intracellular CO2. They investigated the metabolic responses of M. succiniproducens to the different dissolved CO2 concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO2 concentration was below 8.74 mM. The cell growth and succinic acid production increased proportionally as the dissolved CO2 concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO2 concentration of 141 mM were 1.49 and 1.52 times higher respectively, than those obtained at the dissolved CO2 concentration of 8.74 mM. It was also found that the addition of CO2 source provided in the form of NaHCO3, MgCO3, or CaCO3 had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that phosphoenol pyruvate carboxylation by phosphoenol pyruvate carboxykinase is most important for succinic acid production as well as the growth of M. succiniproducens by providing additional ATP.
U.S. Pat. No. 7,223,576 discloses the use of both sodium bicarbonate and gaseous carbon dioxide in the production of succinic acid by a mutant E. coli strain with the heterologous pyruvate carboxylase gene from Lactococcus lactis. The pH of the growth medium was maintained with 1.0 M Na2CO3 and CO2 gas was sparged through the culture during the fermentation period at a constant flow rate. The heterologus expression of pyruvate carboxylase in a succinate producing strain of E. coli increases the carbon flux from pyruvate to oxaloacetic acid. Pyruvate carboxylase diverts pyruvate toward oxaloacetic acid to favor succinate generation.
U.S. Pat. No. 7,244,610 discloses the aerobic succinate production using a bacterial catalyst. The growth medium contained 2 g/L NaHCO3 and approximately 60 mM glucose. NaHCO3 was added to the culture medium because it yielded better cell growth and succinate production due to its pH-buffering capacity and its ability to supply CO2.
U.S. Pat. No. 7,262,046 discloses a growth medium containing 2 g/L NaHCO3 in the aerobic succinate production using a bacterial biocatalysts. The washed culture was then used to inoculate a bioreactor containing LB with 2 g/L NaHCO3.
US Patent Application Publication No. 2006/0073577 A1 discloses the use of LB broth medium supplemented with 20 g/L of glucose, and 1 g/L of NaHCO3 in the production of succinate. NaHCO3 was added to the culture medium because of its pH-buffering capacity and its ability to supply CO2.
US Patent Application No. 2009/0186392 A1 discloses a method of glycerol fermentation where pH and CO2 concentrations are controlled to allow the fermentative metabolism of glycerol to desired chemical precursors. CO2 concentrations were inevitably linked to pH and went down as pH increased because CO2 was converted to bicarbonate. By increasing CO2 to 20-30% the negative effects of increased pH above 7.0 could be reduced. Improved glycerol fermentation was seen with pH 6.3 and 10% CO2, and with pH 7.5 and 20% CO2. Greater concentrations of CO2 were also beneficial.
U.S. Pat. No. 7,256,016 discloses a recycling system for manipulation of intracellular NADH availability. The anaerobic tube experiments were performed using 40 ml or 45 ml glass vials with open top caps and PTFE/silicone rubber septa. Each vial was filled with 35 ml or 40 ml of LB medium supplemented with 20 g/L glucose, 100 mg/L kanamycin, 0 or 50 mM formate and 1 g/L NaHCO3 to reduce the initial lag time that occurs under anaerobic conditions.
In a dual phase growth pattern for production of succinate, the bacterial culture is initially grown in an aerobic condition and transferred to an anaerobic production phase. The succinate production occurs during the anaerobic growth phase. No growth occurs during the anaerobic process. Glucose consumption and product formation rates were essentially constant under anaerobic conditions and the process exhibits a metabolic pseudo-steady-state. The anaerobic biocatalytic process for the production of succinic acid has been shown to consume carbon dioxide under non-growing anaerobic conditions. Since CO2 is incorporated into the carbon backbone as a result of the carboxylation of phosphoenol pyruvate by phosphoenol pyruvate carboxylase, it is hypothesized that different CO2 concentrations in the gas phase would impact the metabolic fluxes and ultimately change the yield and rate of succinate generated. The effect of CO2 on succinate production in dual-phase Escherichia coli fermentation is well documented (Lu et al., 2009).
International patent application WO 2009/083756 A1 published under the Patent Cooperation Treaty provides a large scale microbial culture method for producing succinic acid using a recombinant bacteria containing an over expressed pyruvate carboxylase gene. The culture is initially grown aerobically in a medium devoid of any inorganic carbon. After the growth in the aerobic environment, the bacterial culture is acclimatized to oxygen lean condition wherein the oxygen concentration is brought down to less than 5% oxygen in the reactor by means of purging the with CO2 or CO2 mixed with an inert gas. The carbon dioxide thus supplied provides the source of inorganic carbon required by the pyruvate carboxylase enzyme.
In the experiments with E. coli stain AFP111, it has been shown that when the concentration of CO2 in the gas phase is increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/g·h to 225 mg/g·h and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO2 concentration in the medium, succinate production did not increase further. A four-process explicit model to describe the CO2 transfer and utilization has predicted that at CO2 concentration below about 30-40%, the system becomes limited by gas phase CO2, while at higher CO2 concentrations the system is limited by phosphoenol pyruvate carboxylase enzyme kinetics. At limiting CO2 concentrations, the succinic acid production can be rate limited at different stages. The diffusion of CO2 from the gas phase into the liquid phase may be limiting. As a result of poor equilibrium, the concentration of the CO2 in the liquid phase may be several folds lower than the concentration of CO2 in the gas phase. Another step in the availability of CO2 lies at the transfer of the dissolved CO2 from the exterior liquid phase to the interior of the biocatalysts. The diffusion of dissolved CO2 through the cell membrane may be too slow. Even the permeation of HCO3 through the cell membrane may be insignificant. Once inside the cell, the CO2 is converted into bicarbonate [HCO3−] form so that it can be used as a substrate for the functioning of the phosphoenol pyruvate carboxylase. The conversion of CO2 to bicarbonate is mediated by carbonic anhydrase (Lu et al., 2009).
U.S. Pat. No. 6,455,284 discloses a dual-phase E. coli fermentation for the production of succinic acid. The E. coli strain used in this study contained a polynucleotide sequence encoding a pyruvate carboxylase operatively linked to a promoter, wherein said polynucleotide sequence is expressed and produces an enzymatically active pyruvate carboxylase which is able to incorporate the inorganic carbon in the growth medium into the succinic acid produced. E. coli cells were grown aerobically in Luria-Bertani (LB) medium. Anaerobic fermentation were carried out in 100 ml serum bottles with 50 ml LB medium supplemented with 20 g/L glucose and 40 g/L MgCO3. The fermentations were terminated at 24 hours at which point the pH value of all fermentations were approximately pH 6.7.
US Patent Application Publication No. 2007/0111294 provides growth coupled succinate production in E. coli strains. All experiments were performed using M9 minimal medium at pH 7.0 (6.78 g/L Na2HPO4, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2) supplemented with 2 g/L glucose and 20 mM NaHCO3. The inorganic carbon required for the succinic acid production was provided by NaHCO3 in the medium.
U.S. Pat. No. 7,563,606 provides a method for producing succinic acid using the bacterial strain Brevibacterium flavum MJ-233. Brevibacterium flavum may be currently classified into Corynebacterium glutamicum. These bacterial cells showed an enhanced pyruvate carboxylase activity due to the presence of a plasmid coding for the pyruvate carboxylase activity. The neutralization was carried out by using magnesium carbonate and magnesium hydroxide. Supplementing the magnesium carbonate either with ammonium hydrogen carbonate or sodium hydrogen carbonate enhanced the succinic acid production rate and yield. CO2 gas was also provided to the fermentation vessel. Apparently, the CO2 gas and various carbonate and bicarbonate salts acted as the source of the inorganic carbon required for the action of pyruvate carboxylase enzyme contributing the flow of carbon towards succinic acid.
US Patent Application Publication Nos. 2006/0205048 and 2008/0293113 provide a method for producing succinic acid in a medium containing carbonate ion, bicarbonate ion or carbon dioxide gas and a bacterial strain containing enhanced levels of pyruvate carboxlase enzyme. The suitable bacterial strains are derived from a group consisting of Coryneform bacterium, Bacillus bacterium, and Rhizopium bacterium.
As described above, each of the microbial catalyst currently in use for the production of succinic acid is known to require a source of inorganic carbon for efficient production of succinic acid. In view of the importance of the inorganic carbon in the production of succinic acid, the present invention provides a novel method for preparing solid inorganic carbonate and bicarbonate salts by means of sequestering the carbon released from various industrial applications. The carbon released from fossil fuel burning and the operation of fermentation facilities can be trapped in alkali solutions and the resulting carbonate and bicarbonate salts can be used as a source of inorganic carbon in the fermentative production of succinic acid. In addition, the present invention provides a method for using the product resulting from the sequestration of carbon dioxide.